Nanoliter 2000

Non-viruliferous fourth instar planthoppers were injected with 23 nL of dsRNA of Ubc13 at 6 μg/μL and with 23 nL of 50 μM mouse TNF-α (Sigma-Aldrich, Santa Clara, CA, USA) using Nanoliter 2000 (World Precision Instruments). The insects from the control group were injected with 23 nL of dsRNA of GFP at 6 μg/μL and 23 nL of 20 mM Tris-HCl containing 0.1% Tween 20. Ubc13 transcript levels and activation of JNK were checked after 3 d of treatment. At least three biological replicates and eight insects per replicate were used.

PCR primers with T7 promoter sequences, gps2-dsRNA-F/gps2-dsRNA-R, jnk1-dsRNA-F/jnk1-dsRNA-R, jnk2-dsRNA-F/jnk2-dsRNA-R, ubc13-dsRNA-F/ubc13-dsRNA-R, or TNF-α-dsRNA-F/ TNF-α-dsRNA-R were used to prepare 219-bp double-stranded RNA (dsRNA) of GPS2, 124-bp dsRNA of JNK1, 167-bp dsRNA of JNK2, 142-bp dsRNA of Ubc13, or 107-bp dsRNA of TNF-α (Supplementary file 1). A 420-bp dsRNA for green fluorescent protein (GFP) was amplified using primers gfp-dsRNA-F and gfp-dsRNA-R as negative controls (Supplementary file 1). dsRNA was generated using the T7 RiboMAX Express RNAi System (Promega, Madison, Wisconsin, USA) and purified using Wizard SV Gel and the PCR Clean-Up System (Promega) following the manufacturers’ protocols. Injection of 23 nL of dsRNAs at 6 μg/μL was performed on the fourth instar nymphs. The dsRNAs were delivered into hemolymph in the ventral thorax by microinjection through a glass needle using Nanoliter 2000 (World Precision Instruments, Sarasota, Florida, USA).

Two adult animals were deeply anesthetized by an intramuscular injection of a 3:1 mixture of Ketamine (50 mg/kg, Inresa Arzneimittel GmbH, Freiburg, Germany) and Rompun (20 mg/kg Xylazin, Bayer, Leverkusen, Germany) and were placed in a modified head holder on a stereotaxic frame (Karten and Hodos, 1967 ). Under additional local anesthesia, the skin was incised above the skull, the trabecular bone above the optic tectum was opened, and the descending tecto-tegmental tract at the level of the reticular formation underneath the inferior colliculus was identified using stereotaxic coordinates (Kuenzel and Masson, 1988 ). A micropipette filled with 7.5% (w/v) biotinylated dextran-amine (BDA; MW 3,000; Invitrogen Germany) in phosphate buffer (PB; 0.1 M, pH 7.4) was inserted into the desired location and about 300 nl were injected using a pressure device (Nanoliter 2000, World Precision Instruments, Sarasota, FL, USA). Animals received post-operative treatment and survived for 5–7 days before being sacrificed with an overdose of anesthetics, and subsequent transcardial perfusion with 0.9% normal saline followed by an ice-cold solution of 4% paraformaldehyde (PFA) in PB. Brains were extracted and fixed in 4% PFA in PB for a minimum of 24 h.

APP/PS1 mice were anesthetized with xylazine/ketamine (11.6/73 mg/kg, i.p) and placed in a stereotaxic frame (RWD Life Science) with a mouse adapter. The tip of a pulled glass pipette was inserted at stereotaxic coordinates for AP −1.94 mm, for ML- ±1.5 mm and for DV- −1.0 mm, relative to the bregma, according to our previously report [10 (link),15 (link)]. Viral Vector suspension in a volume of 0.5 μL of PBS buffer was microinjected into the bilateral lateral parietal association (LPtA) cortex of APP/PS1 genotype mice using 2 μL bursts from a Nanoliter 2000 injector (World Precision Instruments). This corresponded to 2 × 10¹² viral genome copies (vgc) of pAAV-MCS-Ppargc1α-m-FLAG-HA and 1 × 10¹² vgc of pAAV-MCS-FLAG-control; expression plasmid was provided by Applied Biological Material co (abm, Zhenjiang, China). It was reported that AAV-mediated transgenic protein expression peaked after three weeks and remains at stable levels after infusion [16 (link),17 (link)]. Accordingly, all behavioral tests and the subsequent molecular examinations were performed three weeks later after mice were infused with AAV. Virus expression was corroborated by immunofluorescence and western blot.

AAV vector solution was microinjected into the right lumbar (L) 4 and L5 DRG using previously described techniques [22 (link)]. Briefly, the surgically exposed intervertebral foramen was slightly enlarged by the removal of laminar bone. Injection was performed through a micropipette that was advanced ~ 100 μm into the ganglion. Rats received L4 and L5 DRG injections of either AAV6-3.2iPA or AAV6-3.2NP (one vector per rat), consisting of 2 μl with adjusted titers containing a total of 2.0 × 10¹⁰ genome viral particles for each DRG. Injection was performed over a 5-min period using a microprocessor-controlled injector (Nanoliter 2000, World Precision Instruments, Sarasota, FL, USA). Removal of the pipette was delayed for an additional 5 min to minimize the extrusion of the injectate. Following the injection and closure of overlying muscle and skin, the animals were returned to their housing where they remained as the designed experiments required.